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1.
Miranda SR Noguti J Carvalho JG Oshima CT Ribeiro DA 《Journal of molecular histology》2011,42(2):181-186
The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis.
For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution
through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay
modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised
purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen
exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas
induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event
during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk
of oral cancer, such as in smokers or alcohol consumers. 相似文献
2.
Eliana Maria Minicucci Glenda Nicioli da Silva Daniel Araki Ribeiro Daisy Maria Favero Salvadori 《Journal of molecular histology》2009,40(1):71-76
The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The
present study aimed to investigate mutations in exon 2 of gene p16CDKN2A during rat tongue carcinogenesis induced by 4-nitroquinoline
1-oxide (4NQO) using direct DNA-sequencing method. A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide
(4NQO) in drinking water for 4, 12, and 20 weeks at 50 ppm dose. Ten animals were used as negative control. No histopathological
changes in tongue epithelia were observed among controls or in the group treated for 4 weeks with 4NQO. Following 12-week
treatment, hyperplasia and epithelial dysplasia were found in mild and moderate forms. At 20 weeks, the tongue presented moderate
and/or severe oral dysplasia and squamous cell carcinoma, with squamous cell carcinoma in the majority of animals. No mutations
were found in any experimental period evaluated that corresponded to normal oral mucosa, hyperplasia, dysplasia and squamous
cell carcinomas. Taken together, our results suggest that p16CDKN2A mutations in exon 2 are not involved in the multistep
tongue carcinogenesis of Wistar rats induced by 4NQO. 相似文献
3.
This study was undertaken to investigate the genomic instability on blood cells during 4-nitroquinoline 1-oxide (4NQO)-induced
rat tongue carcinogenesis by means of single cell gel (comet) and micronucleus assays. Male Wistar rats were distributed into
three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks.
Ten animals were used as negative control. Although no histopathological abnormalities were induced in the epithelium after
4 weeks of carcinogen exposure, genetic damage was found in blood cells as depicted by the mean tail moment and an increase
of micronucleated polychromatic erythrocytes. After 12 and 20 weeks treatment, the same picture occurred, being the strong
effect observed in the micronucleus induction. These periods correspond to pre-neoplastic lesions and well-differentiated
squamous cell carcinomas, respectively. Taken together, our results support the idea that genomic instability on blood cells
appears to be associated with the risk and progression of oral cancer, being a reliable tool for detecting early systemic
conditions of malignancy. 相似文献
4.
Martins C Cação R Cole KJ Phillips DH Laires A Rueff J Rodrigues AS 《Mutation research》2012,747(1):86-92
We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts. 相似文献
5.
Esther Fernández-Miñano Clara Ortiz Ascensión Vicente José L. Calvo Antonio J. Ortiz 《Biometals》2011,24(5):935-941
Although the metal devices used in orthodontic treatments are manufactured highly resistance to corrosion, they may still
suffer some localized corrosion resulting from the oral cavity conditions. The corrosion causes the release of metals from
the alloys used for their manufacture. In this report, we evaluated the in vivo metal ions release of three alloys (stainless
steel, titanium and nickel-free) usually used in the orthodontics treatments and its genotoxicity. We applied to 15 patients,
between 12 and 16 years, 4 tubes and 20 brackets. Samples from oral mucosa were taken before the treatment and 30 days later.
The concentration of the titanium, chromium, manganese, cobalt, nickel, molybdenum and iron were detected using inductively
coupled plasma mass spectrometry (ICP-MS). The genotoxicity was measured with a comet assay (Olive moment). The oral mucosa
cells in contact with the stainless steel alloy displayed the greatest titanium and manganese concentrations and those in
contact with the nickel-free alloy presented the greatest concentration of chromium and iron. Both alloys, stainless steel
and nickel-free, induced a higher DNA damage in the oral mucosa cells than the titanium alloy, in which the Olive moment was
similar to controls. Based on the results of our study, we can conclude that titanium brackets and tubes are the most biocompatible
of the three alloys. 相似文献
6.
Arvind Kumar Nikhlesh Kumar Singh Pushpalata Rabindra Sinha 《Molecular biology reports》2010,37(3):1373-1376
Epidemiologic and experimental studies suggest that the probiotic organisms are effective in preventing colon carcinogenesis,
which is the major cause of mortality and morbidity in western countries. Keeping this in view, a curd (a common Indian fermented
milk product) was prepared by the addition of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and curd culture Lactococcus lactis biovar. diacetylactis. In present study, we have evaluated the anti tumor effect of probiotic curd by monitoring the DNA damage through comet assay.
The rats were allocated to four groups, first group was DMH control group, second group was probiotic curd group in which
probiotic curd was given along with DMH (1,2-dimethylhydrazine) injection, third group was normal curd group in which normal
curd was given along with DMH injection and fourth group was normal control group. Animals received subcutaneous injection
of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected
at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. A significant reduction in
DNA damage (54.7%) was observed in probiotic curd group as compared to DMH control group (88.1%). The probiotic curd was effective
to significantly reduce the L:W ratio in comparison to DMH control group and normal curd. The results of present study show
the protective effects of probiotic curd against DMH induced genotoxicity in colonic cells. 相似文献
7.
Toyoizumi T Ohta R Kawakami K Nakagawa Y Tazura Y Kuwagata M Noguchi S Sui H Yamakage K 《Mutation research》2012,743(1-2):42-51
We have already found that the in vivo skin comet assay is useful for the evaluation of primary DNA damage induced by genotoxic chemicals in epidermal skin cells. The aim of the present study was to evaluate the sensitivity and specificity of the combined in vivo skin comet assay and in vivo skin micronucleus (MN) test using the same animal to explore the usefulness of the new test method. The combined alkaline comet assay and MN test was carried out with three chemicals: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). In the first experiment, we compared DNA- and chromosome-damaging effects of 3 [72, 24 and 3 hours (h) before sacrifice] and 4 applications (72, 48, 24 and 3h before sacrifice) of 4NQO, which induces dermal irritancy. The animals were euthanized and their skin was sampled for the combination test. As a result, the 4-application method was able to detect both DNA- and chromosome-damaging potential with a lower concentration; therefore, in the second experiment, MNNG and B[a]P were topically applied four times, respectively. The animals were euthanized, and then their skins were sampled for combination tests. In the alkaline comet assay, significant differences in the percent of DNA (%DNA) in the tail were observed in epidermal skin cells treated with MNNG and B[a]P. In the MN test, an increased frequency of MN cells (%MN) cells was observed by treatment with MNNG; however, there were no significant increases. In contrast, significant differences in %MN were observed by treatment with B[a]P. From these results, we conclude that the combined in vivo skin comet assay and in vivo MN test was useful because it can detect different genotoxicity with the same sampling time and reduce the number of animals used. 相似文献
8.
Ana Carolina Cuzzuol Fracalossi Sandra Regina Miranda Celina Tijuko Fujiyama Oshima Marcello Franco Daniel Araki Ribeiro 《Journal of molecular histology》2010,41(1):19-25
Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis,
wound healing, angiogenesis, inflammation, and cancer. The purpose of this study was to characterize the role of MMPs as depicted
by the expression of MMP-2 and MMP-9 during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Male Wistar rats were
distributed into three groups of 10 animals each and treated with 4-nitroquinoline 1-oxide solution at 50 ppm through their
drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced
in the epithelium after 4 weeks of carcinogen exposure; however, immunoexpression of MMP-2 was noticed. The same picture occurred
to MMP-9, in which positive expression was detected for this immunomarker. MMP-2 and MMP-9 showed positive expression either
in pre-neoplastic lesions at 12 weeks following carcinogen exposure or in well-differentiated squamous cell carcinoma induced
after 20 weeks of treatment with 4NQO. Taken together, our results support the belief that MMP-2 and MMP-9 play important
role during malignant transformation and conversion of oral mucosa as assessed by immunohistochemistry. 相似文献
9.
DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR 总被引:8,自引:0,他引:8
Lankoff A Krzowski Ł Głab J Banasik A Lisowska H Kuszewski T Góźdź S Wójcik A 《Mutation research》2004,559(1-2):131-142
The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes following treatment with microcystin-LR and ionizing radiation. Our results indicate that MC-LR has an inhibiting effect on the repair of radiation-induced damage. 相似文献
10.
Mary N. Mohankumar S. Janani B. Karthikeya Prabhu P. R. Vivek Kumar R. K. Jeevanram 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2002,520(1-2):179-187
DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens. 相似文献
11.
In mammalian cells it has previously been observed that low DNA-repair activity is correlated wtih high chromosome-aberration frequency. Since fish cells typically express comparatively low amounts of DNA repair, the chromosome aberration test holds potential as a sensitive fish genotoxicity assay. A comparison of in vitro DNA-repairm activity showed HF > CHO > Ul-H = Ul-F following exposure to MNNG and 4NQO. Although peak chromosome-aberration frequency varied CHO > Ul-H > HF, at comparable mutagen concentrations the relationship was Ul-H > HF > CHO following 4NQO exposure and Ul-H > HF = CHO after MNNG exposure. Analyzing for chromosome aberrations at high mutagen concentrations was not possible due t mitotic inhibition/toxicity which varied according to the mutagen and cell line. Micronuclei frequency varied CHO > Ul-H > HF = Ul-F. In CHO and Ul-H, a 10–15 fold increase over the controls compares with only a 2–3 fold increase for HF and Ul-F. These differences are likely related, in part, to the cell-division rate of each line and the coincident repair of the damaged DNA. Reasons for the lack of negative correlation between DNA repair and chromosomal damage in fish cells are discussed. 相似文献
12.
The comet assay for DNA damage and repair 总被引:9,自引:0,他引:9
Collins AR 《Molecular biotechnology》2004,26(3):249-261
The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks
in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids
containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling
comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA
breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward
the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination
with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity
and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that
recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet
tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining
at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing
specific damage. 相似文献
13.
Because of its characteristics, the comet assay has been used to evaluate the ability of virtually any type of eukaryotic
cell to repair different kinds of DNA damage, including double and single strand breaks and base damage. The ability to detect
excision repair sites using the alkaline version can be enhanced by the inclusion of repair inhibitors, DNA synthesis inhibitors,
or chain terminators. In this sense, we evaluated the ability of hydroxyurea (HU) and cytosine arabinoside (Ara-C), for detecting
lesions produced by the alkylating agents ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) in three different
cell systems. Two hundred cells for experimental point were analyzed in the alkaline version of the comet assay, and the results
are evidences of the utility of the assay to detect alkylation of bases in the cells lines MRC-5 and TK-6, as the treatment
with HU +Ara-C significantly increases both the basal and induced frequency of DNA damage. The use of whole blood, although
it detected the effects of MMS, with and without repair inhibitors, failed to detect the effect of the selected dose of EMS
and does not permit detection increases in the background level. 相似文献
14.
The most used animal models in oral cancer research are the hamster treated by dimethylbenzanthracene (DMBA), and the rat treated by 4-nitroquinoline 1-oxide (4NQO). The purpose of this study was to compare the DMBA-induced hamster tongue carcinogenesis and 4NQO-induced rat tongue carcinogenesis by means of morphological analysis. Male Wistar rats were distributed into three groups of ten animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. A total of 18 Syrian golden hamsters were submitted to 0.5% DMBA (dissolved in acetone) topical application three times/week for 4, 12 and 20 weeks. The primary histopathological change i.e., hyperplasia and hyperkeratosis, was evidenced after 4 weeks treatment with DMBA. Regarding 12 weeks treatment, 4NQO and DMBA were able to induce morphological changes as depicted by hyperplasia and dysplasia. At 20 weeks, squamous cell carcinoma was found in the majority of animals for both carcinogens used. Taken together, our results suggest that the hamster experimental model disclosed aspects related with tongue carcinogenesis in lesser time than rats. Probably, such discrepancies depend strongly on route of administration and the susceptibility with respect to animal species. 相似文献
15.
Simon Neumann Andy Reuner Franz Brümmer Ralph O. Schill 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2009,153(4):425-429
In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 ± 1.98% DNA in tail, compared to 0.44 ± 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 ± 6.41% of DNA was found in the comet tail. After ten months, 23.66 ± 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation. 相似文献
16.
Katrin Wischermann Petra Boukamp Peter Schmezer 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2007,630(1-2):122-128
The comet assay is one of the well-accepted tests to measure radiation-induced DNA damage. The most commonly used protocols require single-cell suspensions that are embedded in agarose in order to perform electrophoresis. For adherently growing cells such as human HaCaT skin keratinocytes this method bears several problems. We show that trypsinization required for maintaining single-cell suspensions is prolonged after UV radiation and thereby reduces cell viability and allows partial repair, with the consequence of reduced damage detection after irradiation. Therefore, we here introduce a modified version of the comet assay where HaCaT cells are seeded onto comet slides 24 h before the assay and overlaid with agarose immediately after irradiation. Using this modification we are now able to reproducibly measure high DNA-damage levels (13-fold increase compared with controls) following irradiation with 60 J/cm2 UVA as well as a dose-dependent increase of DNA damage after 10, 20 and 60 J/cm2 UVA. Thus, by maintaining the cells in their natural configuration, i.e. adherently growing, we exclude several artefacts that are likely to influence the damage responses. These include: (i) trypsinization-dependent changes in cell morphology and polarity (clear lateral, i.e. adherent, and apical side of keratinocytes) which are likely of consequence for the gene-expression pattern, (ii) trypsin- and dislodgement-induced damage reducing cell viability, and (iii) the time delay between damage induction and damage evaluation to unpredictable results due to partial repair. Since these advantages pertain to all adherently growing cells, this improved protocol is not restricted to HaCaT cells but offers great potential also with all non-haematopoietic cells for obtaining accurate results and for studying repair processes in a highly reproducible manner. 相似文献
17.
We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects. 相似文献
18.
Amy Wang John L. Robertson Steven D. Holladay Alan H. Tennant Andrea J. Lengi S. Ansar Ahmed William R. Huckle Andrew D. Kligerman 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2007,634(1-2):51-59
Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin–EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA–protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4 h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells. 相似文献
19.
To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster. 相似文献
20.
Günter Speit Thomas Witton-Davies Worapa Heepchantree Kristina Trenz Heike Hoffmann 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2003,542(1-2):33-42
The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells. 相似文献